Identificación molecular y relaciones evolutivas de Pomacea nobilis, base para la autenticación específica del churo negro de la Amazonia peruana / Molecular identification and evolutionary relationships of Pomacea nobilis, basis for the specific authentication of the black apple snail from Peruvian Amazonia

Resumen En la Amazonia Peruana los caracoles dulceacuícolas de la familia Ampullariidae son conocidos como churos y originalmente han sido descritas para Perú alrededor de 20 especies. Aunque son muy usadas para alimentación, medicina tradicional y objeto de muchos estudios para su cultivo e industrialización, solamente es mencionada en la literatura la especie Pomacea maculata. Se llevó a cabo la identificación molecular sobre la base del marcador mitocondrial COI, de individuos de churos negros (Pomacea) comercializados en los mercados de Iquitos, así como los usados en platos a la carta en la ciudad de Lima, contrastados con otros individuos de procedencia de su hábitat natural. Se encontró que estos especímenes expendidos corresponden a la especie Pomacea nobilis (Reeve, 1856). El análisis filogenético molecular mostró que P. nobilis es especie hermana de P. guyanensis, en el grupo de P. glauca, distantemente relacionada de P. maculata. Las distancias no corregidas encontradas entre ellas, para el marcador mitocondrial COI, fueron de 11.33% a 13.17%, mientras que con P. maculata fueron de 13.67% a 15.33%. Estos resultados demostraron la eficacia del código de barras de ADN para la identificación y autenticación de la especie, lo que le da un valor agregado para su eventual comercio de exportación.

Abstract In the Peruvian Amazon, freshwater snails of the Ampullariidae family are known as churos, and around 20 species have originally been described for Peru. Although they are widely used for food, traditional medicine and the object of many studies for their cultivation and industrialization, only the species Pomacea maculata is mentioned in the literature. Molecular identification was carried out based on the mitochondrial marker COI of individuals of "churo negro" apple snails (Pomacea) commercialized in the markets of Iquitos, as well as those used in restaurant dishes in the city of Lima, and contrasted with specimens from their natural habitat. It was found that these specimens, correspond to the species Pomacea nobilis (Reeve, 1856). The molecular phylogenetic analysis showed P. nobilis as the sister species of P. guyanensis, in the P. glauca group, distantly related to P. maculata. The uncorrected distances found between them, for the mitochondrial marker COI, were from 11.33% to 13.17%, while with P. maculate were from 13.67% to 15.33%. These results demonstrated the effectiveness of the DNA barcode for the identification and authentication of the species, which gives it added value for its eventual export trade.

Multiloci analyses suggest synonymy among Rhomboplites, Ocyurus and Lutjanus and reveal the phylogenetic position of Lutjanus alexandrei (Lutjanidae: Perciformes)

Lutjanidae comprises 21 genera and 135 species widespread throughout Atlantic, Indian and Pacific oceans. Nonetheless, the phylogenetic relationships of Lutjaninae remain uncertain. Furthermore, phylogenetic hypotheses for Lutjanus alexandrei, an endemic species from northeastern Brazilian coast, in Lutjanidae are absent so far. Therefore, we carried out multiloci analyses, combining both mitochondrial and nuclear DNA sequences in Lutjaninae species from Western Atlantic focusing on the controversial relationships among Lutjanus, Rhomboplites, and Ocyurus. Besides, we determined the phylogenetic position and dated the origin of L. alexandrei. The phylogenetics trees based on the 4.4 kb for 11 species corroborated the synonym among Lutjanus and the putative monotypic genera. For the dating of L. alexandrei, another nucleotide dataset (3.0 kb; 40 species) validated the genetic identity of this species that diverged from the sister taxon L. apodus between 2.5 - 6.5 Mya, probably as a result of the barrier caused by the muddy outflow from Orinoco and Amazon rivers along the coastal zone. This report is the most robust multiloci analysis to confirm the synonymy of the three genera of Lutjaninae from Western Atlantic and the first reliable inference about the phylogenetic relationships and origin of L. alexandrei.(AU)

A Família Lutjanidae compreende 21 gêneros e 135 espécies, distribuídas ao longo dos oceanos Atlântico, Índico e Pacífico. As relações filogenéticas dos Lutjaninae são incertas. Além disso, a espécie Lutjanus alexandrei, endêmica da costa nordeste do Brasil, não foi inclusa em nenhuma hipótese filogenética até o presente. Assim, realizamos uma análise integrando DNA mitocondrial e nuclear para espécies de Lutjaninae do Atlântico Ocidental, direcionada para a controversa relação entre Lutjanus, Rhomboplites e Ocyurus. Além disso, alocamos filogeneticamente L. alexandrei e datamos sua origem. As árvores filogenéticas baseadas em 4.4 kb de 11 espécies corroboraram a sinonímia entre os monotípicos e Lutjanus. Para a datação de L. alexandrei, outro banco de nuclueotídeos foi analisado (3.0 kb; 40 espécies), validando geneticamente a espécie e a colocando como irmã de L. apodus, da qual se separou entre 2.5 - 6.5 Mya, o que provavelmente foi provocado pela faixa enlameada na região costeira, influenciada pelas descargas dos rios Amazonas e Orinoco, que funciona como barreira. Este trabalho representa a mais robusta análise multiloci direcionada para a sinonimização dos três gêneros de Lutjaninae e a primeira hipótese filogenética a propor um posicionamento e origem para L. alexandrei.(AU)

Description of a new species of Moenkhausia (Characiformes: Characidae) from the upper Paraguay basin, Central Brazil, with comments on its phylogenetic relationships

A new species of Moenkhausia is described from tributaries of the upper rio Sepotuba, Paraguay basin, Brazil. The new species is distinguished from its congeners by a combination of characters, including an inconspicuous oval-shaped vertically elongated humeral blotch, extending horizontally from third through five lateral-line scales, and vertically from third row above lateral line to first row below it, followed by a diffuse field of dark chromatophores in the flank, combined with a well-defined dark line at the base of the anal fin. Furthermore, the phylogenetic position of the new species is presented based on molecular data, showing a close relationship among species of Moenkhausia and Hemigrammus that have a well-defined dark line at the base of the anal fin. Until this moment, this species is only known from in the upper rio Sepotuba basin.(AU)

Uma nova espécie de Moenkhausia é descrita nos afluentes do rio Sepotuba, bacia do Paraguai, no Brasil. A nova espécie se distingue dos seus congêneres por uma combinação de caracteres, incluindo uma mancha umeral discreta de forma oval, alongada verticalmente, que se estende horizontalmente da terceira a quinta escamas da linha lateral e, verticalmente, da terceira fila de escamas acima da linha lateral até a primeira fila abaixo da linha lateral; seguida por escassos cromatóforos no flanco, combinado com uma linha escura bem definida na base da nadadeira anal. Além disso, a posição filogenética da nova espécie é apresentada com base em dados moleculares, mostrando um relacionamento próximo entre as espécies de Moenkhausia e Hemigrammus que possuem uma linha escura bem definida na base da nadadeira anal. Até o momento, essa espécie é conhecida apenas da bacia do Alto Sepotuba.(AU)

FILOGENIA DE ESPECIES DEL SUBGENERO Parides (LEPIDOPTERA: PAPILIONIDAE) BASADA EN SECUENCIAS DEL GEN CITOCROMO OXIDASA I / Species phylogeny of the Subgenus Parides (Lepidoptera: Papilionidae) Based in Sequences of Citochrome Oxidase I Gene

Parides Hübner es el taxón terminal de Troidini, un grupo de mariposas aposemáticas diversificado en el trópico y subtrópico, y modelos de varios complejos miméticos batesianos y mullerianos. Varias de las especies americanas de Parides son simpátricas e involucran poblaciones con variaciones intraespecíficas en los patrones de coloración, lo que genera confusiones en la definición del estatus taxonómico, especialmente en Colombia, punto de convergencia de las biotas de Norte y Suramérica. Este trabajo genera una aproximación a la filogenia de este grupo de mariposas y establece una definición más robusta de algunos de los taxones. Para ello se analizaron ejemplares pertenecientes a 15 taxones del subgénero americano Parides ( Parides ) como grupo interno y se utilizó como grupo externo especies de otros dos géneros estrechamente relacionados de Troidini. Para la extracción del ADN se utilizó el protocolo de Pascual et al. (1997) y DNeasy Kit. Se amplificó el fragmento final del gen Citocromo Oxidasa I (COI) de 476 pb. Para obtener una hipótesis filogenética se realizaron análisis de máxima parsimonia y se evaluó el soporte de cada nodo mediante Jackknife y soporte absoluto de Bremer. También se realizó un análisis bayesiano. La hipótesis resultante sugiere que el subgénero Parides es un grupo parafilético. Molecularmente se hicieron también válidas una especie y cinco subespecies. Los ejemplares analizados de Parides se dividieron en tres grupos principales coincidentes con los grupos Lysander (grupo 1) y Aeneas (grupos 2 y 3) de Rothschild y Jordan (1906).

Parides Hübner is a terminal taxon of Troidini, an aposematic butterfly group that is diverse in the tropics and subtropics, and a model of mullerian and batesian mimetic complexes. Several American species of Parides are sympatric and include populations with intraspecific variation in color pattern, thus creating confusion on their taxonomic status, mainly in Colombia where the biota of North and South America converge. This work presents a phylogenetic hypothesis of these butterflies and proposes a more robust definition of some taxa. For this, 15 taxa of the subgenus Parides were analyzed as ingroup; species of other two genera of Troidini, closer to Parides , were used as out-group. DNA was extracted using the Pascual et al. (1997) protocol and Quiagen DNAeasy kit. A terminal fragment of Cytochrome Oxidase I gen (476 bp) were amplified. We obtained a phylogenetic approximation using maximum parsimony and evaluated the branch support with Jackknife and absolute Bremer support. We also conducted a bayesian analysis. The resulting phylogenetic hypothesis suggested that Parides is a paraphyletic group; the molecular evidence support one species and five subspecies. The analyzed taxa were divided in three principal groups coincident with the Lysander (group 1) and Aeneas (groups 1 and 2) groups proposed by Rothschild and Jordan (1906).

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) on stromata of Cyttaria hariotii in northwestern Patagonian Nothofagus forests

The occurrence and distribution of Xanthophyllomyces dendrorhous associated with Cyttaria hariotii parasitizing three Nothofagus species (N. dombeyi, N. antarctica and N. pumilio) in northwestern Patagonia (Argentina), as well as the factors that may affect this distribution were herein studied. Between 2000 and 2007, samples were obtained from 18 different locations. Based on physiological tests and morphological characteristics of sexual structures, 72 isolates were identified as X. dendrorhous. Representative strains were studied by MSP-PCR fingerprinting and sequence analysis of the ITS region. MSP-PCR fingerprints were similar for the newly isolated strains, and were also identical to the profiles of the strains previously found in this region. Patagonian strains appear to be a genetically uniform and distinct population, supporting the hypothesis that the association with different host species has determined genetically distinct X. dendrorhous populations worldwide. X. dendrorhous was recovered from N. dombeyi and N. antarctica. Approximately half the sampling sites and samples were positive for X. dendrorhous, but the isolation recovery rate was low. X. dendrorhous was absent in the early stages of ascostromata maturation, becoming more abundant in later stages. The present work represents a step forward in the understanding of the natural distribution and ecology of this biotechnologically relevant yeast.

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) asociado a estromas de Cyttaria hariotii en bosques de Nothofagus en el noroeste de la Patagonia. Se estudió la ocurrencia y la distribución de Xanthophyllomyces dendrorhous asociado a Cyttaria hariotii en tres especies de Nothofagus (N. dombeyi, N. antarctica y N. pumilio) del noroeste de la Patagonia (Argentina), y los factores que podrían afectar esta distribución. El muestreo se realizó entre 2000 y 2007 en 18 sitios diferentes. Según las pruebas fisiológicas y las características morfológicas de las estructuras sexuales, 72 de los aislamientos obtenidos se identificaron como X. dendrorhous. Se estudiaron cepas representativas mediante la técnica de MSP-PCR fingerprinting y secuenciación de la región ITS. Los perfiles de MSP-PCR fueron similares, tanto entre los nuevos aislamientos como entre estos y los de cepas previamente obtenidas en la región. Aparentemente, las cepas patagónicas forman una población genéticamente uniforme y distinta de otras poblaciones. Esto apoya la hipótesis de que la asociación con diferentes especies hospedadoras ha determinado la diferenciación genética de X. dendrorhous en todo el mundo. X. dendrorhous se recuperó de N. dombeyi y de N. antarctica. Aproximadamente la mitad de los sitios de muestreo y de muestras fueron positivos para X. dendrorhous, pero la tasa de aislamiento fue muy baja. X. dendrorhous está ausente en estadios tempranos de maduración de ascostromas y se hace más abundante en estadios más tardíos. El presente trabajo contribuye al mejor entendimiento de la distribución natural y la ecología de esta levadura, de relevancia biotecnológica.

ITS-rDNA phylogeny of Colletotrichum spp. causal agent of apple Glomerella leaf spot / Filogenia por ITS-rDNA de Colletotrichum spp., agente causal da mancha foliar da Gala em macieira

Several diseases have affected apple production, among them there is Glomerella leaf spot (GLS) caused by Colletotrichum spp. The first report of this disease in apple was in plants nearby citrus orchards in São Paulo State, Brazil. The origin of this disease is still not clear, and studies based on the molecular phylogeny could relate the organisms evolutionarily and characterize possible mechanisms of divergent evolution. The amplification of 5.8S-ITS (Internal Transcribed Spacer) of rDNA of 51 pathogenic Colletotrichum spp. isolates from apples, pineapple guava and citrus produced one fragment of approximately 600 bases pairs (bp) for all the isolates analyzed. The amplified fragments were cleaved with restriction enzymes, and fragments from 90 to 500bp were obtained. The sequencing of this region allowed the generation of a phylogenetic tree, regardless of their hosts, and 5 isolated groups were obtained. From the "in silico" comparison, it was possible to verify a variation from 93 to 100 percent of similarity between the sequences studied and the Genbank data base. The causal agent of GLS is nearly related (clustered) to isolates of pineapple guava and to the citrus isolates used as control.

A produção de maçã vem sendo comprometida pela ocorrência de muitas doenças, entre as quais se destaca a Mancha Foliar de Glomerella (MFG), causada por Colletotrichum spp. O primeiro relato dessa doença em maçã foi registrado em plantas próximas a pomares de citrus no Estado de São Paulo, Brasil. A origem da MFG ainda não está bem clara, e estudos baseados na filogenia permitirão relacionar o organismo evolutivamente, possibilitando caracterizar possíveis mecanismos divergentes de evolução. A amplificação da região 5.8S-ITS (espaçador interno transcrito) do rDNA de 51 isolados de Colletotrichum patogênicos em de maçã, goiabeira serrana e citrus produziu um fragmento de aproximadamente 600 pares de bases (pb) para todos os isolados analisados. Os fragmentos amplificados foram digeridos por enzimas de restrição, sendo obtidos fragmentos de 90 a 500pb. O sequenciamento dessa região permitiu a construção de uma árvore filogenética com a distribuição dos isolados em cinco grupos, independentemente de seus hospedeiros. A partir da comparação in silico, foi possível verificar uma variação de 93 a 100 por cento de similaridade entre as sequências estudadas e o banco de dados do GenBank. O agente causal da MFG está relacionado (agrupado) a isolados de goiabeira serrana e aos isolados padrões de citrus.

Molecular phylogenetic relationships among four species of the mangrove tree genus Bruguiera (Rhizophoraceae), as revealed by chromosome and RAPD markers

Analysis of karyotype, nuclear DNA content and RAPD markers were performed in four species of Bruguiera (Rhizophoraceae) of Bhitarkanika mangrove forests, Orissa, India. Detailed karyotype analysis revealing 2n=34 in B. cylindrica and 2n=36 in B. gymnorrhiza was reported for the first time and 2n=34 in B. parviflora and B. sexangula was confirmed. On the basis of the common types of chromosomes present among Bruguiera, two distinct groups were found; one consists of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula. The symmetrical karyotype with same chromosome types grouped B. cylindrica and B. parviflora together and presence of Type E chromosomes placed B. gymnorrhiza and B. sexangula in a separate group, suggesting their closer affinity in their respective group. Analysis of chromosome length, volume, INV and 4C DNA content confirmed this division. Nuclear DNA content was two-fold higher (~17.0 pg) in the second group than in the first (~8.0 pg). The amplification products generated through RAPD revealed 1-9 amplicons with size variations from 600 bp to 2 500 bp with 49.31% genetic similarity between B. gymnorrhiza and B. sexangula and 47.10% in between B. cylindrica and B. parviflora. The high copy number marker band (~ 1 100 bp) yielded in OPN-15 primer in B. parviflora the characteristic DNA marker, which was cloned and used as probes for assessment of genetic diversity, and demonstrated its close genetic affinity to B. cylindrica. B. gymnorrhiza and B. sexangula also produced similar marker bands of ~600 bp and ~2 200 bp in the same primer. All of the cytological, 4C DNA content and RAPD data confirmed the existence of two taxonomically distinct groups of Bruguiera: one consisting of B. cylindrica and B. parviflora and the other of B. gymnorrhiza and B. sexangula as placed earlier (1862) in the tribe Rhizophoreae by Bentham and Hooker, on the basis of the flowering habits of Bruguiera. Genetically, the B. sexangula and B. gymnorrhiza group was found to be very closely, rather than distantly, related to B. parviflora and B. cylindrica. Our results demonstrate that molecular markers together with cytological evidence provide an effective tool to access the existing interspecific genetic polymorphism in mangrove species, to solve the taxonomic problems and to design their conservation strategy. Rev. Biol. Trop. 55 (2): 437-448. Epub 2007 June, 29.

Estudiamos cuatro especies del mangle Bruguiera (Rhizophoraceae) en Orissa, India. Los cromosomas indican queB. cylindrica y B. parviflora son un grupo taxonómico, y que B. gymnorrhiza y B. sexangula son otro. Genéticamente, el par B. sexangula y B. gymnorrhiza está cercanamente emparentado con B. parviflora and B. cylindrica. Nuestros datos indican que el uso combinado de marcadores genéticos y evidencia citológica permiten discernir el polimorfismo genético interespecífico en los mangles, tanto para resolver problemas taxonómicos como para diseña estrategias eficaces de conservación.

Relaciones filogenéticas entre géneros de ciervos neotropicales (Artiodactyla: Cervidae) mediante secuenciación de ADN mitocondrial y marcadores microsatelitales

Phylogenetic relationships among Neotropical deer genera (Artiodactyla: Cervidae) by means of DNAmt sequences and microsatellite markers. The current work shows two molecular phylogenetic analyses on Neotropical deers. In the first analysis, the mitochondrial control region (D-loop) was sequenced in six Odocoileinae species from Latin America, using the sequences of two Muntiacinae as outgroups. The results obtained were as follows: A sequence of Mazama americana showed a striking relationship with several sequences of Odocoileus in contrast to that expected, since this M. americana haplotype, from a Mexican origin, was not associated with several Bolivian Mazama sequences analyzed. This could put forward that this genera is not monophyletic. On the other hand, these Bolivian Mazama formed a clade with Pudu puda and Ozotoceros bezoarticus. Likely, an Odocoileus virginianus sequence from the Central area of Colombia showed a more strong relationship with a Northamerican O. heminonus sequence than with the other O. virginianus sequences of Colombian origin as well. This could be explained by means of various different hypotheses. The first is the existence of common ancestral haplotypes between both species. Another one is the reiterative hybridization among both Odocoileus species before the migration of O. virginianus from North America to South America. Moreover, the maximum parsimony analysis showed an intense relationship between the Muntiacinae and this Neotropical Cervidae clade. In addition, and adding credence to the relevant polyphyletism found in Mazama by means of the mitochondrial control region DNA sequences, a second analysis with 16 DNA microsatellite loci also showed a higher genetic relationship between M. americana and O. virginianus, than between the first species regard to Mazama gouazoubira. Rev. Biol. Trop. 55 (2): 723-741. Epub 2007 June, 29.

El presente trabajo muestra dos análisis moleculares sobre la filogenia de los cérvidos neotropicales. En uno se secuenció la región control del mtDNA (D-loop) de seis especies de Odocoileinae, utilizándose las secuencias de dos Muntiacinae como elementos externos. Se evidenciaron los siguientes resultados: La secuencia de una Mazama americana, de origen mexicano, presentó una fuerte relación filogenética con las diversas secuencias estudiadas de Odocoileus, contrario a lo esperado ya que, a priori, debería haberse asociado con las secuencias analizadas de otros ejemplares de Mazama de origen boliviano. Esto pone en evidencia que este género no es monofilético. A su vez, las secuencias de los ejemplares bolivianos de Mazama formaron una agrupación con secuencias de Pudu puda y O. bezoarticus. Una secuencia de O. virginianus, del área central de Colombia, presentó más relación con la secuencia de un O. hemionus norteamericano que con las restantes secuencias analizadas de O. virginianus, también de origen colombiano. Esto puede reflejar varias explicaciones hipotéticas, tales como la existencia de haplotipos ancestrales comunes entre ambas especies de Odocoileus, hasta la hibridación en Norteamérica entre ambos taxones antes de su penetración en Sudamérica. Los análisis de máxima parsimonia presentan una especial relación entre los Muntiacinae y el clado de los ciervos sudamericanos. El segundo análisis filogenético hizo uso de 16 marcadores nucleares microsatélites. Aunque, en principio, estos marcadores no son los más recomendables para estudios filogenéticos intergenéricos, los resultados muestran, al igual que el ADN mitocondrial, una mayor relación entre M. americana y O. virginianus que entre la primera especie y M. gouzaoubira.